Repeated examination and concentration techniques are often required to detect trypanosomes in the blood because parasite numbers vary at different stages of infection. At times no parasite will be found due to immune response. The following techniques are recommended:
- Wet preparation
- Examination of thick stained blood film
- micorhaematocrit concentration techniques
(a) Quantitative buffy coat method (b) Capillary tube method
- Triple centrifugation tube techniques to concentrate trypanosomes when a microhaematocrit centrifuge is not available.
- Miniature anion exchange centrifugation (MAEC) techniques.
- Serological techniques such as: immunofluorescent assay, complement fixation test, enzyme-linked immunosorbent assay, card agglutination test.
If present in high concentration trypanomastigotes can be seen thrashing among the cells on a fresh, unstained wet preparation of blood. However, preparation should be examined within 4 hours of sampling or add few milligrams of glucose to extend the time.
Apply a drop of blood on slide, cover with cover slip and view using x40 objective with iris condenser partially closed.
Examination of thick stained blood film:
The same technique applied in normal thick film is used.
Microhaematocrit Concentration Techniques:
The microhaematocrit technique is rapid and recommended for detecting motile trypanosome in blood when a microhaemmatocrit centrifuge is available. It also enables the packed cell volume (PCV) to be measures to check whether the patient is anaemic.
Procedure of capillary tube method:
- Fill two capillary tubes with capillary blood or two plain capillary tubes with fresh anticoagulated (EDTA) blood to about 10mm from top.
- Seal the dry top end of the tubes using sealant
- Centrifuge the tubes in a microhaematocrit centrifuge for 3-5mins
- Wipe clean the area of the capillary tubes where it will be viewed, i.e. the region between the buffy coat and plasma.
- Mount the two capillary tubes on a slide, supported on two strips of plasticine.
- Fill the space between the two tubes with clean water and cover the cover slip.
- Examine immediately the plasma just above the buffy coat layer for motile trypanosomes. Use a x20 objactive or x10 with iris condenser closed to focus and the x40 objective to confirm that the motility is due to trypanosome.
If present, the trypanosomes will be seen in the region
The preparation must be examines within a few minutes of the blood being centrifuged otherwise the trypanosomes will migrate into the supernatant plasma and be missed.
Quantitative buffy coat method:
The procedure is same as in detection of malaria parasite but after centrifugation the tube is left t stand up right for 5 minutes before viewing microscopically.
Triple centrifugation tube techniques:
The technique is appropriate when a microhaematocrit centrifuge cannot be performed or when microfilariae are also present in the blood.
- Collect 9ml of venous blood into a tube containing 1ml of 6% w/v sodium citrate anticoagulant and mix gently.
- With the minimum of delay, centrifuge the fresh blood at slow speed, i.e. 100g for 10 minutes. Transfer the plasma and cells above the red cells to a centrifuge tube.
- Centrifuge at a slightly higher speed i.e. 200-100g for 10minutes and transfer the supernatant fluid to another tube.
- Centrifuge at 700-900g for 10minutes, remove and discard the supernatant fluid. Tap the end of the tube t resuspend the sediment and transfer the entire sediment to a glass slide, cover with slip.
- Immediately examine microscopically for motile trypanosomes using x40 objectives. If microfilariae are present, allow to dry, the fix in methanol for 2 minutes and then stain with giemsa. Microscopically, the parasites are 13-42mm long with a single flagellum, a centrally placed nucleus and small dot-like kinetoplast.
Miniature Anion Exchange Centrifugation (MAEC) Techniques
This technique is considered by most workers to be the most sensitive for detecting small numbers of T.b. rhodesiense or T.b. ganbiense trypanosomes, but it is an expensive technique and requires careful control. It is mostly found in specialist laboratories.
In the MAEC technique, the patient’s heparinized blood is passed through a buffered anion (negatively charged) exchange column of diethyl amino ethyl (DEAE)-52 cellulose. As the blood I eluted through the column, the strongly charged trypanosomes are washed through the column with the buffered saline. The eluate is then collected, centrifuged and the sediment is examined microscopically for motile trypanosomes. The cellulose column requires careful preparation and the pH of the buffer is critical to ensure absorption of the cells and elution of the trypanosomes.
SEROLOGICAL TECHNIQUES FOR BLOOD PARASITE EXAMINATION:
The principle of the serologic techniques is based on antigen-antibody reaction to give a visible agglutination reaction. Serologic techniques can be applied for mass field survey. These include; immunoflourescent assay, complement fixation test, enzyme-linked immunosorbent assay and card agglutination test.
BLOOD PARASITE: AMERICAN TRYPANOSOMIASIS
American trypanosonomiasis (chagas disease) is caused by Trypanosoma cruzi which is transmitted by the reduvidae bug subfamily triatomiae. Chagas’ disease is only found in tropical and sub-tropical South and Central American Countries.
Mode of transmission:
T. cruzi is mainly transmitted through contact with the feaces of an infected blood sucking bug which contains the infective trypomastigotes that is deposited on skin or mucous membrane as the bug feed from its host.
BLOOD PARASITE EXAMINATION: EXAMINATION OF BLOOD FOR T.CRUZI
Because of the fact that the trypomastigotes of T.cruzi are more fragile that those causing African trypanosomiasis, serology rather than morphology is recommended foe initial screening. Some of the techniques include;
- Careful microscopical examination of fresh blood motile trypanosomes using wet preparation.
- Microhaematocrit concentration technique. This is rapid and sensitive technique.
- Test tube centrifugation concentration technique using clotted sample (strout technique) or lysed venous blood (Hoff Technique)
- Polymerase chain reaction
- Serological tests
OTHER NON-HAEMATOLOGICAL DIAGNOSIS INCLUDES
- Animal inoculation
- Culture method
- Collect 5-10ml of venous blood into a dry glass tube
- Incubate for 1 hour at 37C
- Transfer the serum to a centrifuge tube, cap the tube and centrifuge for 5 minutes at low speed of about 400g.
- Discard the supernatant fluid and examine the sediment microscopically for motile trypanosome using 40x objective.
HOOF CONCENTRATION TECHNIQUE
- Collect 2.5ml of venous blood into EDTA anticoagulant and mix gently.
- Dispense 6-7ml of ammonium chloride lysing solution into a conical centrifuge tube; add the EDTA blood sample and mix.
- Leave for about 3 minutes to allow time for the red cell to lyse. Cap the tube and centrifuge for 15 minutes at low to medium speed of 400-500g.
- Discard the supernatant and examine the sediment microscopically for motile trypanosomes using x40 objectives
POLYMERASE CHAIN REACTION:
This has been used to detect positive patients with few as one trypomastigotes in 20ml of blood. PCR may be very useful for diagnosis of patients with chronic disease because of the lack of sensitivity and specificity of serologic test and lack of sensitivity for xenodiagnosis.